Immobilized carbohydrate biosensor

ABSTRACT

The present invention refers to a biosensor in which an immobolized carbohydrate or a derivative thereof is used to generate a detectable signal when a protein, a virus or a cell is bound to the carbohydrate surface. The sensor is an optical sensor, a piezoelectric sensor, an electrochemical electrode or a thermistor. A method of binding carbohydrates to a gold surface is also described.

This is a continuation application based on U.S. patent application Ser. No. 08/356,229, filed Dec. 12, 1994, now U.S. Pat. Ser. No. 6,231,733, which in turn is a continuation application of PCT/SE94/00343, filed Apr. 18, 1994; which was in turn based on Swedish Priority Document 9301270-6, filed Apr. 7, 1993. All of these documents are relied upon and incorporated by reference herein, in their entirety.

The present invention relates to a biosensor in which a carbohydrate or a derivative thereof is used to generate a detectable signal via the specific binding of a protein, a virus or a cell.

BACKGROUND

Biosensors are characterised by a physical or chemical signal transducer, which response is activated by 8 specific interaction between a biochemical structure (which directly or indirectly has been bound to the transducer) and one or several analytes.

Biosensors are used to detect the analyte/analytes and in certain cases also for quantification of the analyte/analytes.

The advantages of the biosensor are that a physical or chemical transducer has been made specific so that a general physical or chemical parameter (e.g. temperature, pH, optical density) can be used for the detection of one specific substance in a complex mixture of non-specific substances.

The limitations of the biosensor are the specificity of the biochemical structure bound to the transducer, the range of specificity and stability, and, that the transducer signal has to be made independent of the background changes in the parameter that the transducer is measuring. In methods of Enzymology, volume 137, several articles are describing different aspects of biosensors.

Definitions.

Biosensor—physical or chemical signal transducer, e.g. photometer, chemical electrode, temperature or pressure signal transducer, which directly or indirectly has been connected with a biochemical structure. In previous biosensors one has preferentially used an enzyme, a specific protein or antibody as the biochemical structure and in this way the biosensors have been given the property of being able to detect substances which specifically bind to the biochemical structure in a qualitative or quantitative way.

Reflection measurement—measurement of the intensity of light reflected from a surface where the properties of the surface influences the reflection, e.g. biomolecules which change the refraction index of the surface.

Polarisation measurement—measurement of the polarisation of polarised light, usually as the angle of polarisation, which is depending on the binding of biomolecules, virus or cells.

Surface plasmon spectroscopy—optical physical measurement technique which utilise the surface plasmon condition of thin metal surfaces, which can be used to measure small changes of refraction index with high sensitivity, e.g. as caused by the presence of biomolecules on the surface.

Ellipsometry—optical physical measurement technique which can be used to measure small changes of refraction index at surfaces with high sensitivity, by measuring changes in elliptisity of polarised light. e.g. as caused by the presence of biomolecules on the surface.

Piezoelectric crystal—crystal which frequency can be influenced by changes of mass or pressure which can be measured electrically, for example the change of mass caused by the presence of biomolecule(s), virus or cell(s) bound to the crystal surface.

Electrochemical electrode—measuring device which generates an electrical signal caused by an electrochemical reaction at the electrode which is related to a chemical parameter, e.g. pH, pO₂, pCO₂, the values of which can vary because of the presence of analyte(s) in a sample specific for a compound bound to the measuring device.

Thermistor—electrical resistance device which changes resistance with the temperature; biochemical reactions are characterised by e.g. specific values of heat consumption/formation, which can be registered via the thermistor.

A large amount of the carbohydrate sequences present in glycoproteins or in glycolipids, and usually also smaller fragments of these sequences, have shown biospecific binding to proteins, virus or cells.

The present invention describes a biosensor where this specificity is used for determination of such a component in a sample. The invention is characterised by that the carbohydrate or a derivative thereof. Is bound to a surface in the biosensor.

As carbohydrate, one can use fragments (oligosaccharides) of the carbohydrate sequences found in glycoproteins or in glycollpids and one can also use smaller fragments of these sequences, i.e. disaccharide, trisaccharide, tetrasaccharide or a pentasaccharide, because this size usually is sufficient for the oligosaccharide to bind a protein, virus or a cell in a biospecific manner. A review or such carbohydrate sequences can be found in e.g. Chemistry and Physics of Lipids, vol. 42, p. 153-172, 1986, and in Ann. Rev. Biochem., vol. 58, p. 309-350.

The oligosaccharide is usually modified in the reducing end with an aglycon, which is composed of a glycosidically bound organic group which is suitable for binding to the surface in the biosensor. Examples of aglycons are OEtSEtCONHNH₂, —OEtSPhNH₂, etc. The binding to the surface in the biosensor can be done directly or via a proteins, e.g. bovine serum albumine or via a chemical structure which has been adsorbed or which has been covalently bound to the surface. Such a chemical structure can contain reactive organic groups such as carboxyl-, sulfonate, cyanate, epoxy-, aldehyde groups or other groups suitable for chemical conjugation with for example an amine or thiol group in the aglycon.

More specific examples of analytes which can be analysed with biosensor according to the present invention are lectins, antibodies against carbohydrates, pathogenic virus or bacteria, such as urinary tract bacteria (e.g. P-fimbriated E. coli) or pathogens of the respiratory tract, and bacteria which cause infections/diarrhea in in gastrointestinal tract.

Non-limiting examples of carbohydrate structures of interest and which can be used in the form of a carbohydrate derivative in a biosensor according to the invention, are monosaccharides, disaccharides, trisaccharides and higher oligosaccharides which show biological activity or which has the ability to specifically bind one or more biomolecules or a group of biomolecules. Examples of biomolecules are other saccharides, peptides and proteins. Examples of such carbohydrate sequences are the blood group determinants (for example A, B, H, Lewis-a, Lewis-b, Lewis-x, Lewis-y), cancer-associated carbohydrate sequences, carbohydrate sequences (often di-, tri- or tetrasaccharides) which bind to pathogenic bacteria/toxins or virus of for example the respiratory, the gastro-intestinal or the urinary tract, carbohydrate sequences which bind to proteins/cells/white blood cells associated with inflammatory reactions (for example selectin-carbohydrate reactions).

These and other carbohydrate structures which can be used in a biosensor according to the present invention often contain one or more of the following monosaccharides (or a derivative or an analog of any of these) which are α- or β-glycosidically bound: hexosamine, fucose, mannose, glucose, N-acetylglucosamine, N-acetyl-galactosamine, xylose, galactose, or another monosaccharide. These components are usually present in for example pyranose or furanose form.

Examples of carbohydrate derivatives are derivatives where the carbohydrate or a derivative or an analog, are modified in the reducing end with an O-, N-, C- or S-glycosidically bound aglycon which can be an alifatic or an aromatic compound, an amino acid-, peptide- or protein molecule or a derivative thereof. The aglycon part can thus be composed of for example an O-, N-, C- or S-glycosidically bound aliphatic or aromatic compound which is bound to an amino acid-, peptide- or protein molecule or a derivative thereof. Examples of carbohydrate derivatives which can be used according to the invention are structures in which one or more of the hydroxyl groups in the carbohydrate, in addition to or instead of the hydroxyl group in the reducing end of the carbohydrate part, have been modified with an organic or inorganic group. This can be of interest, for example to increase/modify the biological activity or to facilitate the binding to the biosensor surface according to the invention.

The aglycon part or another group can be used for adsorption or covalent binding of the carbohydrate derivative to the surface of the biosensor and can be used in the invention as a spacer molecule between the biosensor surface and the carbohydrate part to minimise sterical hindrance in the binding of the analyte to the carbohydrate part in the biosensor according to the invention.

The aliphatic or aromatic compound in the aglycon can for example consist of structures of the type —R—X, where R- consists of an organic compound, for example an alkyl chain of the type (—CH₂)_(n)—, in which n is an integer, e.g. in the interval 2 to 8, or is composed of an aromatic group-containing structure, and where —X is for example a structure of the type —S—, amide (—NH—CO— or CO—NH—), amine (—NH—), a —N═N— group or another group suitable for binding to the surface in the biosensor or to a protein (i.e. in the latter case the carbohydrate derivative is a neoglycoprotein). When the carbohydrate derivative is a neoglycoprotein R can be used as a spacer between the protein part and the carbohydrate part. The spacer often has a functional part (—X— above) which has been used in the binding to the protein.

Suitable spacer and functional group is chosen by the person skilled in the art and does not limit the scope of the invention.

The carbohydrate derivative can also, according to the invention, be composed of a natural, in vitro isolated glycoprotein or a recombinant glycoprotein or a glycopeptide. This type of derivative can be adsorbed to the surface in the biosensor, for example a gold- or silica surface or another surface which adsorbs proteins, lipids or peptides.

In the case a covalent binding is desired one can, as in the case when the carbohydrate derivative is a neoglycoprotein, use for example the protein part's amino-, carboxyl-, or thiol groups for binding to the surface in the biosensor. This (as for the synthesis of the neoglycoprotein from carbohydrate spacer and protein) can be done with the standard techniques which normally are used for modification of proteins and for immobilisation of proteins to solid supports (see for example methods mentioned in Methods of Enzymology, volumes 44, 102, and 135), and the choice of suitable technology is made by the person skilled in the art in every specific case. Examples of methods are coupling or activation of carboxyl groups with carbodiimide reagents, N-hydroxysuccinimide reagents, of hydroxyl groups with CNBr, sulphonyl chloride (tresyl chloride, tosyl chloride), divinyl sulphone, periodate (gives aldehyde groups), thiol groups are activated with thiol reagents of the type SPDP, etc.

As examples of surfaces according to the invention may be mentioned:

-   Carbohydrate-R—X-Biosensor surface or -   Carbohydrate-R—X-Protein-Biosensor surface -   where Carbohydrate, R and X have been exemplified above. X and     Protein can be directly adsorbed on the Biosensor surface above, but     between X and Biosensor surface above and between Protein and     Biosensor surface above can also a chemical group be present, for     example a —CO—CH₂CH₂—S— group, i.e. for example: -   Carbohydrate-R—NH—CO—CH₂—CH₂-S-Biosensor surface or -   Carbohydrate-R—X-Protein-NH-O—CH₂—CH₂—S-Biosensor surface.

As protein one can use for example bovine serum albumin, but all for the application suitable types of proteins can be used in the carbohydrate derivative-based biosensor according to the invention.

The biosensor according to the invention can be designed in a variety of configurations. Examples are:

-   a) planar carbohydrate surface which easily can be contacted with     the sample, for example a surface designed as a dipstick, this     surface can be placed in a measuring device for optical reflectance     measurement in air. -   b) Flow system with flow cell, the surface of which is modified with     carbohydrate and where the signal is transferred with optical,     electrochemical, thermical or gravimetric method and where the     measuring device is placed in, or in close connection with the cell. -   c) Cuvettte or other sample cell, which has been connected with a     signal transducer equipped with carbohydrate to which the sample is     added. -   d) Planar carbohydrate surface which consists of part of the signal     transducer which with ease can be brought into contact with the     sample for a suitable time, whereafter the sample is removed and the     surface of the signal transducer is characterised with a physical     measuring method, for example electronic measurement, gravimetric     measurement or thermal measurement.

In some situations, e.g. to increase the biosensor signal in the measurement of low concentrations of cells, it can be advantageous in the measurement of the analyte with the biosensor to add, after the binding of the analyte to the carbohydrate surface, microparticles modified with carbohydrate specific for the bound cell.

The surface of the biosensor can be, for example a gold surface or a modified gold surface, a plastic surface which has been modified with a gold surface, sliver surface or another metallic surface, or modifications thereof with polymers to which chemical coupling of carbohydrate can be carried out.

Below are given non-limiting examples of carbohydrate surfaces which can be used in biosensors according to the invention for binding and analysis/determination of pathogenic bacteria of the urinary tract.

EXAMPLE

One example was performed as follows: Silica surface coated with a gold layer was modified with mercaptopropionic acid by dipping the surface in a 5 mM solution of the acid. The carboxyl groups were modified with carbodiimide (EDC) for 2 hours, whereafter digalactoside with aglycon (Galα1-4Galβ-OEtSEtCONHNH₂), was coupled to the EDC-activated surface for 12 hours at pH 8.5 and the surface was then rinsed with buffer.

The thus obtained gold surface modified with digalactoside was dipped for 60 minutes (this time can be varied) in a sample with bacteria of the urinary tract (P-fimbriated E. coli) containing Gal 1-4Gal-specific receptor protein, followed by rinsing of the surface with distilled water for 2 minutes. Another gold surface modified in the same way with Galα1-4Gal, was dipped in a sample containing another non-infectious E. coli strain which lack the Galα1-4Gal-specific receptor protein. The extent of binding of the different bacteria to the surfaces was compared with electrom microscopy. The bacteria with The Galα1-4Gal-receptor bound to the surface to a ca 10-15 times higher extent than the other bacteria. The binding of P-fimbriated E. coli to a gold surface modified with mercaptopropionic acid alone, was ca 20 times lower than to the Galα1-4Gal-modified surface.

Alternative non-limiting examples are given below in which a neoglycoprotein was bound covalently or adsorbed directly on a surface for use in biosensor according to the invention.

In procedure B, Galα1-4GalβOCH₂CH₂SCH₂CH₂C(O)—NHNH-BSA was coupled to the same type of EDC-activated gold plate as in the procedure above. The Galabiose-BSA derivative (0.1 mg/ml) was dissolved in 0.1 M boronate, pH 8.5 and EDC-activated plates were immersed in this solution for 1 hour. Subsequently the plates were immersed in a BSA solution (3 mg/ml) in phosphate buffer for 1 minute and rinsed with buffer and distilled water and stored as above.

In procedure C, Gold plates (not pretreated with mercaptopropionic acid) were immersed in a solution of Galα1-4Galβ-BSA (0.1 mg/ml) in 0.1 M sodium phosphate, pH 6.0, for 1 hour and subsequently immersed in the above BSA solution (3 mg/ml) for 1 minute, rinsed with buffer and distilled water and stored as above.

These latter biosensor surfaces showed similar characteristics and low background binding of bacteria as surface in the first example above. 

1. An immobilized carbohydrate derivative biosensor, comprising: a surface; and a carbohydrate derivative, bound to the surface, which specifically binds to at least one biomolecule in a sample wherein the carbohydrate derivative comprises a fragment of a carbohydrate sequence found in a glycoprotein or a glyco lipid which is an oligosaccharide modified in the reducing end with an aglycon selected from the group consisting of —OEtSEtCONHNH₂ and OEtSPhNH₂.
 2. An immobilized carbohydrate derivative biosensor, comprising: a surface; and a carbohydrate derivative, bound to the surface, which specifically binds to at least one biomolecule in a sample wherein the carbohydrate derivative is a derivative in which the carbohydrate is modified in the reducing end with an O-, N-, C- or S-glycosidically bound aglycon, and said glycosidically bound aglycon comprises a structure corresponding to a formula —R—X, wherein the biosensor corresponds to the formula: carbohydrate-R—NH—CO—CH₂CH₂—S-biosensor surface.
 3. An immobilized carbohydrate derivative biosensor, comprising: a surface; and a carbohydrate derivative, bound to the surface, which specifically binds to at least one biomolecule in a sample wherein the carbohydrate derivative is a derivative in which the carbohydrate is modified in the reducing end with an O—, N—, C- or S-glycosidically bound aglycon, comprising a structure corresponding to a formula —R—X, and wherein the biosensor corresponds to the formula carbohydrate-R—X-protein-NH—CO—CH₂—CH₂—S-biosensor surface wherein X is a member selected from the group consisting of —S—, —NH—CO—, CO—NH—, —NH—, and —N=N—.
 4. The immobilized carbohydrate derivative biosensor according to claim 3, wherein the protein comprises bovine serum albumin.
 5. An immobilized carbohydrate derivative biosensor, comprising: a surface; and a carbohydrate derivative, bound to the surface, which specifically binds to at least one biomolecule in a sample, wherein the surface comprises silica coated with a gold layer modified with mercaptopropionic acid by dipping the surface in a 5 mM solution of the acid.
 6. The immobilized carbohydrate derivative biosensor according to claim 4, wherein carboxyl groups of said acid are thereafter modified with carbodiimide (EDC), whereafter Galα1-4Galβ-OEtSEtCONHNH₂ is coupled to the surface for 12 hours at pH 8.5, and the surface rinsed with a buffer.
 7. A method of using the immobilized carbohydrate derivative biosensor according to claim 6, comprising: dipping the surface in a sample containing bacteria of the urinary tract having Galα1-4Gal-specific receptor protein; thereafter rinsing the surface with distilled water; and determining the extent of binding of the bacteria to the surface.
 8. The immobilized carbohydrate derivative biosensor according to claim 1, wherein the carbohydrate derivative comprises: Galα1-4GalβOCH₂CH₂SCH₂CH₂C(O)—NH H-BSA, wherein BSA is bovine serum albumin.
 9. The immobilized carbohydrate derivative biosensor according to claim 1, wherein the carbohydrate derivative comprises Galα1-4Galβ-BSA, wherein BSA is bovine serum albumin. 